System and method for structured illumination and collection for improved optical confocality of raman fiber array spectral translator imaging and interactive raman probing

ABSTRACT

The disclosure relates generally to methods and apparatus for using a fiber array spectral translator-based (“FAST”) spectroscopic system for improved imaging, spectral analysis, and interactive probing of a sample. In an embodiment, the confocality of a fiber array spectral translator-based spectroscopic system is improved through the use of structured illumination and/or structured collection of photons. User input may be received and acted upon to allow a user to interactively in real time and/or near real time view and analyze specific regions of the sample.

PRIORITY INFORMATION

The instant disclosure claims the filing-date benefit of ProvisionalApplication No. 60/778,588 filed 2 Mar. 2006, entitled “SpectralUnmixing in a Fiber Array Spectral Translator (FAST) Based PolymorphScreening”, the disclosure of which is incorporated herein in itsentirety. The instant disclosure is also related to pending U.S. patentapplication Ser. No. 10/812,233, filed 29 Mar. 2004, entitled “Methodfor Identifying Components of a Mixture via Spectral Analysis” and topending U.S. patent application Ser. No. 11/000,683, filed 20 Nov. 2004,entitled “Multipoint Method for identifying Hazardous Agents”, thedisclosure of each is hereby incorporated by reference in its entirety.All of the foregoing are commonly assigned to the assignee of theinstant disclosure.

BACKGROUND

A fiber array spectral translator (“FAST”) system when used inconjunction with a photon detector allows massively parallel acquisitionof full-spectral images. A FAST system can provide rapid real-timeanalysis for quick detection, classification, identification, andvisualization of the sample. The FAST technology can acquire a few tothousands of full spectral range, spatially resolved spectrasimultaneously. A typical FAST array contains multiple optical fibersthat may be arranged in a two-dimensional array on one end and a onedimensional (i.e., linear) array on the other end. The linear array isuseful for interfacing with a photon detector, such as a charge-coupleddevice (“CCD”). The two-dimensional array end of the FAST is typicallypositioned to receive photons from a sample. The photons from the samplemay be, for example, emitted by the sample, reflected off of the sample,refracted by the sample, fluoresce from the sample, or scattered by thesample. The scattered photons may be Raman photons.

In a FAST spectrographic system, photons incident to the two-dimensionalend of the FAST may be focused so that a spectroscopic image of thesample is conveyed onto the two-dimensional array of optical fibers. Thetwo-dimensional array of optical fibers may be drawn into aone-dimensional distal array with, for example, serpentine ordering. Theone-dimensional fiber stack may be operatively coupled to an imagingspectrograph of a photon detector, such as a charge-coupled device so asto apply the photons received at the two-dimensional end of the FAST tothe detector rows of the photon detector.

One advantage of this type of apparatus over other spectroscopicapparatus is speed of analysis. A complete spectroscopic imaging dataset can be acquired in the amount of time it takes to generate a singlespectrum from a given material. Additionally, the FAST can beimplemented with multiple detectors. The FAST system allows formassively parallel acquisition of full-spectral images. A FAST fiberbundle may feed optical information from its two-dimensional non-linearimaging end (which can be in any non-linear configuration, e.g.,circular, square, rectangular, etc.) to its one-dimensional lineardistal end input into the photon detector.

Given the advantageous ability of a FAST system to acquire hundreds tothousands of full spectral range, spatially-resolved spectra, such asRaman spectra, substantially simultaneously, a FAST system may be usedin a variety of situations to help resolve difficult spectrographicproblems such as the presence of polymorphs of a compound, sometimesreferred to as spectral unmixing.

Chemical images may generally be acquired using one of two classes ofapproaches: (1) scanning, and (2) widefield chemical imaging. Inscanning methods, a radiation source is focused onto the surface of asample and a spectrum from each spatial position is collected using adispersive spectrograph or interferometer. Long data collection timesare common with scanning methods since the duration of the experiment isproportional to the number of image pixels. Because of such long datacollection times, scanned images are captured at low image definition,which relates directly to the limited utility of the technique as animaging tool for the routine assessment of material morphology.Furthermore, the spatial resolution of the image is limited by the sizeof the source illumination on the sample and the rastering mechanism,which requires the use of moving mechanical parts that are challengingto operate reproducibly. In addition, for light-absorbing materials,scanning methods present an enormous challenge. These materials have lowdamage thresholds, requiring the use of low laser power densities tominimize local thermal expansion and sample degradation.

Despite the limitations, scanning methods are relatively maturetechniques and have been applied in a number of applications. Anadvantage of scanning-based chemical imaging is the ability to capturethe entire spectrum in an efficient manner. This advantage is bestrealized in the research evaluation of new material systems where theunderlying spectroscopy is not well understood, and therefore, benefitsmay be available from the analysis of the entire spectrum.

In widefield chemical imaging, the entire sample field of view isilluminated and analyzed simultaneously. Numerous widefield chemicalimaging approaches have been demonstrated, with the majority of methodsinvolving the recording of an image at discrete spectral intervalsthough an imaging spectrometer (i.e., LCTF (Liquid Crystal TunableFilter), AOTF (Acousto-Optic Tunable Filter), etc.).

Because both (X-Y) spatial dimensions are collected simultaneously inwidefield Chemical Imaging using imaging spectrometers, the experimentduration is proportional to the number of spectral channels and not tothe number of image pixels. The widefield advantages are best realizedwhen high fidelity images at a limited number of wavelengths providesufficient chemical and spatial information. In most materialscharacterization applications, only a limited number of spectral bands(typically <100) are required to analyze the analytes of interest. Byreducing the number of spectral channels, the duration of the widefieldexperiment decreases without losing spatial resolution. In addition,time-dependent changes in the sample are only observed in the spectraldimension, which simplifies the analysis of chemical images in widefieldimaging.

Conversely, attempts to reduce the duration of scanning experiments (inthe scanning approach discussed above) compromise either the spatialresolution or the field of view. Reducing the number of spectralchannels in scanning mode has little effect on the experiment durationsince the entire chemical spectrum is captured simultaneously (in thescanning approach discussed above). Scanning experiments record timedependent sample changes as spatial variations. Pixels collected atdifferent times often have induced spectral differences that complicateanalysis.

A limitation that widefield illumination methods suffer from issecondary scattering of illumination that fundamentally reduces theinherent confocality associated with the measurement(s). Secondaryscattering occurs when illumination of a first location results in anemission or scattering of radiation that migrates to a second samplelocation and is detected as if it had originated in the second spatiallocation. On the other hand, the scanning approach discussed above isgenerally less susceptible to secondary illumination effects since theillumination is first restricted to a first sample location and thecollected light is then restricted to the same sample location throughuse of pinhole apertures. Line scanning approaches are slightly moresusceptible to secondary scattering effects along the sample axis thatis aligned in parallel with the entrance slit of the spectrographcompared to point mapping/scanning approaches.

FAST enables full spectral acquisition for hundreds to thousands ofspatially resolved spectra in a single image frame—dramaticallyincreasing data acquisition rates compared to current tunable filterbased technologies. Software is used to extract the spatial/spectralinformation to reconstruct hyperspectral (chemical imaging) data cubesof the original object. Furthermore, FAST is a rugged technology thatoperates over an extensive spectral range from ultraviolet (UV) toinfrared (IR).

As with alternative widefield chemical imaging methods, FAST basedsystems are susceptible to secondary scattering of radiation. Thepresent disclosure describes systems and methods for overcoming thelimitations of the prior art including novel sample illumination andlight collection systems and methods to enhance the confocality of FASTbased spectroscopy systems.

Accordingly, it is on object of the present disclosure to provide animproved FAST system for detecting photons from a sample includingincreasing the confocality of the system by directing only photons in apredetermined group of plural fibers in the FAST system to a photondetector, wherein each fiber in the predetermined group is associatedwith a predetermined different portion of the sample wherein each of thepredetermined different portions is smaller than an illuminated portionof the sample.

It is another object of the present disclosure to provide a system fordetecting photons from a sample, comprising a photon source forilluminating a first portion of a sample with first photons to therebyproduce second photons; a fiber array spectral translator comprisingplural fibers for receiving the second photons and directing the secondphotons to the photon detector, wherein only a first predetermined groupof the plural fibers receive and direct the second photons to the photondetector, and wherein each fiber in the first predetermined group isassociated with a predetermined different second portion of the samplewherein each of the predetermined different second portions is smallerthan the first portion; and the aforementioned photon detector fordetecting the second photons. Furthermore, the system may furthercomprise a display device, and a microprocessor unit operativelyconnected to the photon detector and to the display device, wherein thephoton detector sends to the microprocessor a first signalrepresentative of the second photons, and wherein the microprocessorunit forms a second signal from the first signal and sends the secondsignal to the display device to display a first visual representation ofthe second signal.

It is a further object of the present disclosure to provide a method fordetecting photons from a sample, comprising illuminating a first portionof a sample with first photons to thereby produce second photons;receiving the second photons using a fiber array spectral translatorcomprising plural fibers and directing the second photons to the photondetector using the fiber array spectral translator, wherein only a firstpredetermined group of the plural fibers receive and direct the secondphotons to the photon detector, and wherein each fiber in the firstpredetermined group is associated with a predetermined different secondportion of the sample wherein each of the predetermined different secondportions is smaller than said first portion; and detecting the secondphotons using the photon detector.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic block diagram of a of a fiber array spectraltranslator (“FAST”) based spectroscopy system.

FIG. 2 is a is a schematic drawing of a FAST based spectroscopy system.

FIG. 3 is a schematic drawing of a FAST fiber layout showing anexemplary spatial mapping arrangement.

FIGS. 4A through 4H illustrate details of an exemplary FAST basedspectroscopy system according to one embodiment of the disclosure.

FIGS. 5A through 5D illustrate different structured illuminationarrangements in a FAST based spectroscopy system according toembodiments of the disclosure.

FIGS. 5E through 5H illustrate different structured illumination andcollection arrangements in a FAST based spectroscopy system according toembodiments of the disclosure.

FIGS. 6A through 6D illustrate a test sample (FIG. 6A) and graphs (FIGS.6B-6D) showing data collected from the test sample using a FAST basedspectroscopy system according to an embodiment of the disclosure.

FIGS. 7A through 7C illustrate a test sample (FIG. 7A), a comparison ofdata using FAST single fiber and FAST multiple fibers (FIG. 7B), and agraph (FIG. 7C) showing data collected from the test sample using a FASTbased spectroscopy system according to an embodiment of the disclosure.

FIGS. 8A through 8D illustrate a test sample (FIG. 8A) and graphs (FIGS.8B-8D) showing data collected from the test sample using a FAST basedspectroscopy system according to an embodiment of the disclosure.

FIGS. 9A through 9F illustrate a method using a FAST based spectroscopysystem according to one embodiment of the disclosure for real-time ornear real-time user-interactive generation of spectra from one or morespatial locations on a sample.

FIG. 10 is a block diagram of a FAST based spectroscopic system withoptional user input according to one embodiment of the disclosure.

FIG. 11A is a block diagram of a FAST based spectroscopic system withoptional user input according to one embodiment of the disclosure.

FIG. 11B is a block diagram of a FAST based spectroscopic system withoptional user input according to another embodiment of the disclosure.

FIG. 12 is a flow chart of a method for detecting photons using a FASTbased spectroscopic system according to an embodiment of the disclosure.

FIG. 13 is a flow chart of a further method for detecting photons usinga FAST based spectroscopic system and displaying a visual representationof the detected photons according to an embodiment of the disclosure.

FIG. 14 is a flow chart of a further method for detecting photons usinga FAST based spectroscopic system, displaying a visual representation ofthe detected photons, and selecting a portion of the visualrepresentation based on user input according to an embodiment of thedisclosure.

FIG. 15 is a flow chart of a further method for detecting photons usinga FAST based spectroscopic system, displaying a visual representation ofthe detected photons, storing a second visual representation in a memorydevice, and overlaying the two visual representations according to anembodiment of the disclosure.

DETAILED DESCRIPTION

An emerging technology in the field of widefield chemical imaging is theuse of fiber optic arrays. Briefly, FIG. 1 illustrates a block diagramof an exemplary Fiber Array Spectral Translator (“FAST”)-basedspectroscopy system. FIG. 2, on the other hand, provides a more detailedarchitectural view of the FAST system illustrated in FIG. 1. A FASTsystem may also be referred to as Dimension Reduction Arrays. FIG. 3illustrates a simplified, exemplary, arrangement of optical fibers in aFAST fiber bundle having a two-dimensional (“2D”) imaging end and aone-dimensional (“1D”) distal end for feeding photons into a photondetector.

With reference now directed toward the various figures, FIG. 1illustrates a block diagram of an exemplary FAST-based spectroscopysystem including a spectrometer/detector 101, a FAST fiber bundle 102,an imaging lens 103, a collecting lens 104, a stage 105 for holding,e.g., a 96-well plate containing samples which may be a mixturecontaining polymorphs of a compound, a photon source 106, such as thelaser shown, and a control unit 107 for controlling thespectrometer/detector 101, the photon source 106 and the stage 105. FIG.2, on the other hand, provides a more detailed architectural view of theFAST system illustrated in FIG. 1. In FIG. 2, the system may include aspectrometer/detector 201, a FAST fiber bundle 202, which may bearranged in a substantially circular 19-fiber arrangement as shown incross-sectional view 202 a, a lens 203, which may be an imaging lens, alens 204, which may be a collecting lens, sample 205 which may bemounted in a well of a well plate and positioned on a stage, such as thestage 105 described above with respect to FIG. 1, a photon source 206,which may be a laser as shown, a control unit 207, which may control thespectrometer/detector 201, the laser 206, and the sample 205, a filter208 which may be a 0° filter such as a laser rejection filter, a filter209 which may be a 7° filter, such as a laser rejection filter, a lens210, which may be a focusing lens, and a mirror 211. A FAST system mayalso be referred to as a Dimension Reduction Array since, in anembodiment, the imaging end may be a 2D array and the distal end may bea 1D array. FAST technology can acquire hundreds to thousands of fullspectral range, spatially resolved spectra, such as Raman spectra,simultaneously. This may be accomplished by focusing an image onto a twodimensional array of optical fibers (at the end of the fiber bundlewhich is proximal to the sample to be viewed) such as the FAST fiberbundle 202 which may be drawn into a one dimensional distal array (atthe end of the fiber bundle which feeds the optical signals into thespectrometer/spectrograph, i.e., where the FAST fiber bundle 202 entersthe spectrometer/detector 201) with serpentine (or curvilinear) orderingas illustrated in the exemplary embodiment in FIG. 3. The onedimensional fiber stack may be coupled to a spectrometer/detector 201such as an imaging spectrograph. Software and/or hardware may thenextract the spectral/spatial information that is embedded in a singleCCD image frame.

Referring now to FIG. 3, the construction of the FAST array requiresknowledge of the position of each fiber at both the imaging end and thedistal end of the array as shown, for example, in the simplified diagramfor FIG. 3 where a total of sixteen fibers are shown numbered incorrespondence between the imaging (or proximal) end 301 and the distalend 302 of the fiber bundle. As shown in FIG. 3, a FAST fiber bundle mayfeed optical information from its 2D non-linear imaging end 301 (whichcan be in any non-linear configuration, e.g., circular, square,rectangular, etc., and may contain more than the 16 fibers shown in theexemplary embodiment in FIG. 3) to its 2D linear distal end 302, whichfeeds the optical information into associated detector rows 303. Thedistal end may be positioned at the input to a photon detector 303,which may include a spectrometer/spectrograph and a CCD, a complementarymetal oxide semiconductor (“CMOS”) detector, or a focal plane arraysensor (such as InGaAs, InSb, HgCdTe (“MCT”), etc.). Photons exiting thedistal end fibers may be collected by the various detector rows. Eachfiber collects light from a fixed position in the two-dimensional array(imaging end) and transmits this light onto a fixed position on thedetector (through that fiber's distal end).

FIG. 3 shows a non-limiting exemplary spatial arrangement of fibers atthe imaging end 301 and the distal end 302. Additionally, as shown inFIG. 3, each fiber may span more than one detector row in detector 303,allowing higher resolution than one pixel per fiber in the reconstructedimage. In fact, this super-resolution, combined with interpolationbetween fiber pixels (i.e., pixels in the detector associated with therespective fiber), achieves much higher spatial resolution than isotherwise possible. Thus, spatial calibration may involve not only theknowledge of fiber geometry (i.e., fiber correspondence) at the imagingend and the distal end, but also the knowledge of which detector rowsare associated with a given fiber.

Thus, in an exemplary FAST application, a fiber bundle may be physicallyorganized in 2D (X-Y) at the signal input end so as to image the samplein two dimensions. On the output side, however, the fibers in the fiberbundle may be stacked in a linear or curvilinear array (1D) (principallyX or Y direction only depending on the slit placement) and aligned witha slit in the grating-based spectrometer so as to facilitate extractionof spectral info. It is known that a spectrometer works on a liner (1D)input. This 1D output from the fiber bundle may be fed to thespectrometer gratings (or other similar dispersive elements) to separatesignal wavelengths. Each wavelength-dispersed signal (1D) from thegratings may be sent to the CCD detector as shown in the extremelysimplified view of FIG. 3. Each column of CCD pixels may represent onewavelength. There may be 5 CCD pixels (or rows) mapped to an image point(or fiber) at a particular wavelength, for example. Thus, in the case of1024 pixels in a column, around 204-205 (1024 divided by 5) image points(or linear fiber array outputs) can be accommodated. A 1D-to-2D arraymapping may then organize each column of CCD back to or close to theoriginal 2D fiber bundle arrangement so as to obtain the 2D image of thesample for the specific wavelength (also known as a 3D spectral image).

The FAST-based chemical imaging method may provide a significant speedof analysis. Using FAST, a complete chemical imaging data set can oftenbe acquired in approximately the amount of time it takes to generate asingle spectrum from a given material with a conventional non-FASTmethod. Fusion of FAST-generated chemical images and high-spatialresolution images generated using other modalities can providesignificant insight into the morphology and chemistry of materials.Furthermore, a FAST system may provide significant instrumentation costreduction, expanded free spectral range (UV-IR), and optionalsensitivity to polarization.

FAST enables full spectral acquisition for hundreds to thousands ofspatially resolved spectra in a single image frame—dramaticallyincreasing data acquisition rates compared to current tunable filterbased technologies. Software and/or hardware may be used to extract thespatial/spectral information to reconstruct hyperspectral (chemicalimaging) data cubes of the original object. Furthermore, FAST is arugged technology that operates over an extensive spectral range (fromUV to IR).

In the FAST optical system embodiment of FIG. 2, a two-lens imagingconfiguration is shown, although the present disclosure is not limitedto such a configuration, as would be obvious to those of skill in theart. The system in FIG. 2 may include a collecting lens 204, an imaginglens 203, and some optics (e.g., filters 208 and 209, described above)for laser illumination for spectroscopy, such as Raman spectroscopy. Thecollecting lens 204 may be a doublet for focusing the laser beam ontothe sample and collecting photons from the sample, such as Ramanradiations/Raman scattered photons from the sample. The collecting lens204 may also collimate the imaging beams (e.g., the Raman photons) andproject images in infinity. The imaging lens 203 may also be a doubletand may be selected in such a way that when it is used together with thecollecting lens 204, images, e.g., of Raman radiations, will be formedat its final focal plane. Because the imaging beams between thecollecting lens 204 and the imaging lens 203 are collimated, it may beeasier to introduce one of more laser filters, such as filters 208and/or 209, into the FAST optics as shown in FIG. 2.

In one embodiment of the present disclosure, the FAST system of FIG. 1may be used to screen or detect polymorphs present in a sample (e.g., a96-well plate, referred to above as stage 105 in FIG. 1). The detectionmay be accomplished by matching spectra of the observed target sampleagainst a set of library spectra. Thus, in case of a mixture containingpolymorphs, a spectrum of a polymorph crystal may be matched against aset of library spectra of various polymorphs to identify the polymorphor polymorphs present in the mixture. In one embodiment, the libraryspectra of a plurality of known polymorphs of a compound may bepre-stored electronically (e.g., in a computer memory used along withthe FAST system of FIG. 1, as shown in FIG. 10 discussed below). Suchspectra may have been obtained in a device-independent manner (i.e., thespectra may not be taken using the FAST system selected for currentpolymorph screening application). In an alternative embodiment, thelibrary spectra may be generated using the same FAST system as thatbeing used for current polymorph screening application at hand. Hence,in such an embodiment, the library spectra may be device-dependent and,hence, may be matched more accurately with the target polymorph spectra.

In one embodiment, there may be 19 fibers in the fiber bundle. As willbe obvious to those of skill in the art, the present disclosure is notlimited to a 19-fiber FAST bundle and can be implemented with any numberof fibers in the FAST bundle in any type of 2D orientation at theproximal, or imaging, end. The fiber bundle may be sequentially focusedon each well in the 96-well plate placed on the stage 105 of FIG. 1. Thestage 105 may be designed to receive samples for spectroscopic analysis.Each well may contain a plurality of polymorphs, in which case theresulting spectrum may be a combination of individual polymorph spectra.Various spectral matching techniques may be employed to identify whichknown polymorphs are present in the well being investigated. Also, thosespectra that do not match with the library spectra may indicate presenceof unknown polymorphs in the sample at hand. Such information may beuseful in further analyzing the sample for detection and identificationof such new polymorphs.

With reference now directed toward FIGS. 4A-4G, details of an exemplaryFAST based spectroscopy system according to one embodiment of thedisclosure are illustrated. As discussed above, FAST technology canacquire hundreds to thousands of full spectral range, spatially resolvedRaman spectra simultaneously. This may be accomplished by focusing animage from a sample (FIG. 4A showing regions 401 a, 402 a, and 403 a )using a light gathering optic (FIG. 4B) onto a two dimensional array ofoptical fibers (FIG. 4C showing regions 401 c, 402 c, and 403 c whichcorrespond to regions 401 a, 402 a, and 403 a, respectively) such as aFAST bundle, that may be drawn into a one dimensional distal array withstructured (i.e., serpentine or curvilinear) or unstructured (i.e.,random) ordering (FIG. 4D). The one dimensional fiber stack may becoupled to a dispersive spectrograph (FIG. 4E) which may be connected toa detector, such as the CCD shown. Software, hardware, or a combinationof the two may then extract the spectral/spatial information that isembedded in a single CCD image frame (FIG. 4F showing regions 401 f, 402f, and 403 f which correspond to regions 401 a, 402 a, and 403 a,respectively) to produce spatial-specific spectra (FIG. 4G showingregions 401g, 402 g, and 403 g which correspond to regions 401 a, 402 a,and 403 a, respectively) and/or spectral-specific images (FIG. 4Hshowing regions 401 h, 402 h, and 403 h which correspond to regions 401a, 402 a, and 403 a, respectively) which may be displayed on anappropriate display device (e.g., a computer screen, a television,etc.). As shown in FIG. 4G, the spectral-specific spectra may be a CCDrow extraction for spectral reconstruction. A 1D-to-2D array mapping maythen organize each column of CCD information back to or close to theoriginal 2D fiber bundle arrangement so as to obtain the 2D image of thesample for the specific wavelength (also known as a 3D spectral imageand illustrated in FIG. 4H). As shown in FIG. 4H, the spatial-specificimage may be a CCD column extraction for image reconstruction.Additionally, the display may include both a spectral reconstruction andan image reconstruction. Fiber array based chemical imaging has beendemonstrated in several applications including Raman chemical imaginganalysis of microcomposites and biomaterials and time-resolved atomicemission chemical imaging of laser-induced plumes.

FIGS. 5A through 5H show some exemplary uses of FAST for improvedconfocality for use in spectroscopic systems, such as for widefieldchemical imaging. A sample shown schematically in FIG. 5A includingregions 501 a, 502 a, and 503 a may be illuminated globally (FIG. 5B),i.e., an entire area of the sample (or the entire sample) isilluminated, illuminated in a point-focused manner (FIG. 5C) where onlyone point or region of the sample is illuminated, in FIG. 5C region 502Cis the only illuminated region of the sample, or randomly (FIG. 5D)where only the three regions 501 d, 502 d, 503 d of the sample areilluminated. Regions 501‘x’, 502‘x’, and 503‘x’ throughout FIGS. 5Athrough 5H, where ‘x’ represents ‘a’ through ‘h’, are correspondingregions, respectively. Returning to FIG. 5C, the region 502 c is theonly illuminated region of the sample and this may be achieved numerousways including structured fiber optic illumination using a FAST-basedspectroscopic system with or without the use of optical lenses. In anembodiment, regions 501 a, 502 a, and 503 a may represent threeexemplary fibers in a fiber bundle of a FAST system (e.g., the FASTsystem of FIG. 1). It is observed here that the optical confocality of ameasurement may be improved when combined with the use of FAST asdiscussed herein.

In FIG. 5E, the illumination of and collection of light from the sampleis achieved with through the same fiber. In other words, theillumination and collection optics is the same—the fibers in the singlefiber bundle in the FAST system—in the embodiment of FIG. 5E.Specifically, FIG. 5E shows a 30 fiber FAST bundle, such as the oneillustrated in FIG. 4C, where the illuminating light travels througheach of the 30 fibers to illuminate the sample and each of the 30 fibersreceives light from the sample and directs that received light to, forexample, a photon detector. An embodiment of the disclosurecontemplates, but the disclosure is not limited to, a situation wherethe illumination region and the collection region for any one fiber ismutually exclusive of the illumination region and the collection regionof the other fibers in the FAST bundle. In the configuration shownschematically in FIG. 5F, the sample is globally illuminated (with anillumination source, e.g., an angled laser as shown in FIG. 2 or via adispersive fiber) that is different from the light collection mechanism(i.e., one or more fibers in the fiber bundle of the FAST system) andlight is gathered with all fibers within the FAST bundle. In FIG. 5G,the illumination is restricted to a small area around region 502 g andthe light is gathered from an individual fiber (e.g., the fiberrepresented by the circle 502 g in FIG. 5G) or a smaller number offibers consistent with the geometry and size of an object of interest inthe sample. The illumination in FIG. 5G may be accomplished using alaser as shown, for example, in FIGS. 1 and 2, or using one or morefibers in the fiber bundle of the FAST (in which case the illuminationsource and the light collection source may be the same). In FIG. 5H, theillumination is structured and restricted to areas of interest (i.e.,501 h, 502 h, and 503 h) within the sample while the collected radiationis primarily captured by a restricted number of fibers in the FASTbundle (corresponding to areas 501 h, 502 h, and 503 h). In theembodiment of FIG. 5H, the structured illumination optics may include alaser coupled with an optical switch or a pattern creation optics toaccomplish the desired structured illumination. The structuredillumination can be accomplished either sequentially or simultaneously(i.e., in parallel). It is noted here that various illumination andcollection approaches illustrated in FIGS. 5A through 5H may be part ofa non-destructive imaging system of, for example, a chemical orbiological sample.

FIGS. 6-8 show real data collected using a prototype FAST systemconfigured as shown schematically in FIG. 5F. A multilayer polymerstandard (shown schematically in FIG. 6A) with known layer thicknesseswas analyzed depth-wise using a FALCON™ Raman chemical imaging systemmarketed by Chemlmage Corporation of Pittsburgh, Pa., wherein theFALCON™ system was equipped with a 64-fiber FAST bundle. Spectra atvarious sample depths were acquired using the FAST bundle and compared.The spectra from the light collected from all fibers were compared tothe spectra obtained from a single fiber (FIGS. 6B-6D). In each of theFIGS. 6B-6D, the top spectrum in the respective graph is obtained usinga FAST single fiber and the bottom spectrum is obtained using the entireFAST bundle of 64 fibers. The axes of the graphs in FIGS. 6B-6D RamanShift in wavenumbers for the horizontal axis and Intensity (in arbitraryunits) for the vertical axis. It is observed here with reference to FIG.6A that the multilayer polymer standard included the following layers ofmaterials: the top layer 601 is Polyethylene Terephthalate (“PET”), thenext layer down 602 is an adhesive layer following the PET layer, themiddle layer 603 is an Ethylene Vinyl Alcohol (“EV-OH”) copolymer, thenext layer down 604 is another adhesive layer following the EV-OH layer,and the bottom layer 605 is Low Density Polyethylene (“LD PE”) andTitanium Dioxide (“TiO₂”). The thicknesses of the layers are shown inFIG. 6A.

As is known in the art, the depth analysis represented in FIGS. 6-8 maybe accomplished by obtaining a spectrum at a specific layer of thepolymer standard and then moving the stage (item 105 in FIG. 1) holdingthe sample containing the polymer standard sample upward so as to enablethe collecting optics to image the next successive layer. It is notedthat using a single fiber from FAST effectively limits the area/volumefrom which the light is gathered. The data collection from the singlefiber was accomplished by restricting the rows from which data wascollected from the spectrometer CCD as discussed later hereinbelow withreference to FIG. 9. As can be seen in each of FIGS. 6B (PET), 6C(EVOH), and 6D (LDPE&TiO2) that the peaks in the spectra for the singleFAST fiber case (spectra 606 b, 606 c, and 606 d) are generally thinnerthan the peaks in the spectra for the 64 FAST fiber case (spectra 607 b,607 c, and 607 d), which indicates that the single FAST fiber case hasan increased confocality over the 64 FAST fiber case.

Referring now to FIGS. 7A-7C, FIG. 7A illustrates the same multilayerpolymer standard illustrated in FIG. 6A which includes the followinglayers of materials: the top layer 701 is Polyethylene Terephthalate(“PET”), the next layer down 702 is an adhesive layer following the PETlayer, the middle layer 703 is an Ethylene Vinyl Alcohol (“EV-OH”)copolymer, the next layer down 704 is another adhesive layer followingthe EV-OH layer, and the bottom layer 705 is Low Density Polyethylene(“LD PE”) and Titanium Dioxide (“TiO₂”). The thicknesses of the layersare shown in FIG. 7A. FIG. 7B exemplifies a false-color, depth-wisereconstruction of the FAST data comparing the two modes of detection(single fiber 706 vs. the entire 64-fiber bundle 707) where the uppersample surface (i.e., the top surface of the PET layer 701) is at thetop of the reconstruction and the bottom of the reconstruction is 36microns below the upper sample surface (as measured by movement of thestage upon which the sample rests). FIG. 7C shows spectra depictingMultivariate Curve Resolution (MCR) pure component estimates for PET(701), EVOH (703), and LDPE+TiO₂ (705) layers from the data obtained inthe single fiber mode of detection 706. As can be seen from FIG. 7B, theFAST single fiber case shows much sharper distinction between the layersthan the FAST 64 fiber bundle case. Therefore, the FAST single fibercase allows for more specific readings throughout the sample than theFAST 64 fiber bundle case.

Referring now to FIGS. 8A-8D, FIG. 8A illustrates the same multilayerpolymer standard illustrated in FIG. 6A which includes the followinglayers of materials: the top layer 801 is Polyethylene Terephthalate(“PET”), the next layer down 802 is an adhesive layer following the PETlayer, the middle layer 803 is an Ethylene Vinyl Alcohol (“EV-OH”)copolymer, the next layer down 804 is another adhesive layer followingthe EV-OH layer, and the bottom layer 805 is Low Density Polyethylene(“LD PE”) and Titanium Dioxide (“TiO₂”). The thicknesses of the layersare shown in FIG. 8A. FIGS. 8B-8D show Raman intensity plots for threelayers differing in composition in the multilayer polymer system of FIG.8A. The plots in FIGS. 8B-8D are as a function of sample depth (movementof the stage on which the sample rests) in microns as the horizontalaxis with a normalized intensity value as the vertical axis. Thelayer-specific wavelength is also indicated along with the respectivelayer material in FIGS. 7B-7D. The reduced full-width at half maximum(“FWHM”) plot for the FAST single fiber (e.g., FWHM=8 in FIG. 8C) whencompared to the 64 fibers (e.g., FWHM=9 in FIG. 8C) is an indication ofthe enhanced confocality that the FAST single fiber measurementprovides. It is anticipated for configurations in which the illuminationis structured (e.g., as shown in FIGS. 5A-5D) in addition to thestructured collection (e.g., as shown in FIGS. 5G-5H) thatfurther/additional confocality improvement may be achieved since thestructured illumination/structured collection case further restricts theillumination and sampling volumes of the sample.

FIGS. 9A through 9F illustrate a further method of FAST for enabling areal-time or near real-time user-interactive means of generating spectraassociated with one or more spatial locations within an image. FIG. 9Aillustrates a sample including regions 901 a, 902 a, and 903 a. A FASTimage is gathered from the sample using a plurality of fiber optics(FIG. 8B). In one embodiment, the illumination and collection in FIG. 9Bis similar to the approach depicted in FIG. 5F and described above. Inanother embodiment, the illumination and collection in FIG. 9B may beaccomplished using the approach depicted in FIG. 5E and described above.The fiber optics within the FAST bundle are addressable (in software,hardware, and/or a combination of the two) to respective spatiallocations on the sample and image data from the fibers are capturedusing one or more detectors. In one embodiment, each fiber in the fiberbundle may be linked (or mapped) through software, hardware, or acombination of software and hardware with a respective spatial locationor region in the sample (e.g., regions 901 c, 902 c, and 903 c, whichcorrespond to regions 901 a, 902 a, and 903 a, respectively), and thespectral data collected by each fiber may be captured by that row(s) inthe CCD detector which may be associated with that specific fiber. Inone embodiment, a user may select one or more spatial locations from acomputer-displayed image of the sample with the use of a computerkeyboard, mouse and/or touch screen (as represented by the large arrowin FIG. 9D selecting region 903 d). A computer-displayed optical imageof the sample may be generated using a video camera or CCD-based imagingdevice (different from the CCD detector with the spectrograph) (notshown) that may be placed along the optical path of collection optics torecord an optical image of the sample as is known in the art. Because ofthe known spatial linking between various sample regions andcorresponding fibers, and availability of information on association ofvarious CCD rows with their respective fibers, the operative software,hardware, or combination of software/hardware may be then configured tocollect data from only that portion of the FAST detector image (FIG. 9E)which is associated with the spatial location(s) selected by the user.Referring to FIGS. 9B-9F, fiber #7 in the FAST bundle (FIG. 9B) isselected by the user (FIG. 9D) such that the associated row in the CCD(FIG. 9E) is displayed on an imaging device (FIG. 9F). Such selective orstructured collection of spectral data allows the software, hardware, orcombination of software/hardware to generate a representative spectrum(or spectra) of only the user-selected spatial location(s) asillustrated by spectrum 903 f in FIG. 8F. Thus, although the sample maybe globally illuminated and although each fiber in the fiber bundle(which is a 30-fiber bundle in the exemplary embodiment of FIG. 9B as inthe embodiment of FIG. 4C) may be collecting optical information fromthe sample and sending that information to the CCD detector, thesoftware, hardware, or combination of software and hardware may beconfigured to collect (from the CCD detector) only that spectralinformation which is associated with the sample spatial location/regionselected by the user. Such user-interactive spectrum/spectra (e.g.,Raman spectra) generation may facilitate real-time or near real-timeprobing of sample regions of interest.

In one embodiment, such selective spectrum generation approach may beextended to include overlaying (not shown) the optical image (generatedusing, e.g., a video camera) with a false-colored FAST image (e.g., aRaman chemical image) that is generated based on a correlation betweenthe spectral signatures produced in real-time with FAST and a library ofknown spectral signatures. This allows the user to visualize the samplechemistry in real-time.

With reference now directed towards FIG. 10, an exemplary systemaccording to an embodiment of the present disclosure is illustrated inblock diagram form. A photon source 1001 may illuminate with firstphotons a sample 1002, which may contain polymorphs of a compound, tothereby produce second photons. The photon source 1001 may be anytypical photon source used for spectrographic purposes, such as a laser,white light source, UV (ultraviolet) lamp, etc. A fiber array spectraltranslator 1003, having plural fibers receives the second photons anddirects them to a photon detector 1004 which is operatively connected tothe fiber array spectral translator. The photon detector 1004 mayinclude a dispersive spectrograph (not shown) or other similar equipmentas is known in the art. The photon detector 1004 detects the secondphotons to thereby obtain a first spectrum. A microprocessor unit 1005is operatively connected to the photon detector 1004 and to a memoryunit 1006. The memory unit 1006 may store a set of second spectra whereeach spectrum of the set of second spectra may be representative of adifferent polymorph of the compound (sample 1002). The microprocessorunit 1005 may compare the first spectrum with the set of second spectrato thereby determine the presence of one or more polymorphs in themixture based on said comparison. A display unit 1007 may be operativelyconnected to the microprocessor unit 1005 for displaying spectra and/orimages generated from the photons detected by the photon detector 1004.Optionally, the microprocessor 1005 and/or the display unit 1007 may beadapted to accept user input, such as via a computer mouse or pointingdevice, a keyboard, or, in the case of the display unit 1007, atouch-screen. The user input, as described above, may include userselection of specific information for display of specific spectra and/orimages.

FIGS. 11A and 11B show further embodiments of systems as described aboveand with respect to FIG. 10, like reference numbers refer to likedevices. FIG. 11A illustrates an exemplary system for a single FASTfiber illumination and collection arrangement where the output of thephoton source 1101 is directed to a source rejection filter 1108, suchas a laser rejection filter, dichroic beamsplitter, or similar device,such that the illuminating photons rejected by the filter (i.e.,reflected) are focused onto a single fiber of the 1D end of a FASTbundle. The illuminating photons are then delivered to the sample 1102at the 2D end of the FAST bundle where they, for example, interact withthe sample 1102 to generate, second (e.g., scattered) photons which arethen gathered by the same fiber in the FAST bundle and directed towardsthe photon detector 1104. FIG. 1 B illustrates a further embodimentwhich includes a first lens 1109, a second lens 1110, and a lensletarray 1111. In this embodiment, the rejected (i.e., reflected) photonsfrom the photon source 1101 are focused onto a single fiber of the 1Dend of a FAST bundle via lens 1109. The illuminating photons aredelivered to the sample 1102 at the 2D end of the FAST bundle. A lensletarray 1111 may be coupled to the 2D end of the FAST array for improvedefficiency in delivering and collecting the illuminating photons and thesecond (e.g., scattered, or Raman scattered) photons. The collectedsecond photons (e.g., reflected/scattered source light, scattered lightfrom sample, emitted light from sample, etc.) are collimated by lens1109 and filtered by the source rejection filter 1108. The photonsemitted/scattered by the sample that pass through the source rejectionfilter 1108 are focused onto the entrance slit of a spectrograph/photondetector 1104 via lens 1110.

FIG. 12 is a flow chart of a method for detecting photons using a FASTbased spectroscopic system according to an embodiment of the disclosure.At block 1201, a sample is illuminated with first photons to producesecond photons. In an embodiment, the second photons may be fluorescentphotons from the sample without the need to illuminate the sample withthe first photons. At block 1202, the second photons are received at the2D end of a FAST bundle and the second photons are directed towards aphoton detector (which may typically include a spectrograph, as is knownin the art) where the second photons are detected by the photon detectorat block 1203.

FIG. 13 is a flow chart of a further method for detecting photons usinga FAST based spectroscopic system and displaying a visual representationof the detected photons according to an embodiment of the disclosure.Blocks 1301, 1302, and 1303 correspond to blocks 1201, 1202, and 1203,respectively, as described above. At block 1304, a signal is sent by thephoton detector to a microprocessor unit. The signal may berepresentative of the second photons detected by the photon detector. Atblock 1305, the microprocessor unit may then form a second signal basedon the received first signal and, at block 1306, send the second signalfrom the microprocessor unit to a display device. Alternatively, themicroprocessor unit may simply relay the signal from the photon detectorto the display device with little or no change to the signal from thephoton detector. At block 1307, the display device may display a visualrepresentation of the signal from the microprocessor unit (e.g., thesecond signal).

FIG. 14 is a flow chart of a further method for detecting photons usinga FAST based spectroscopic system, displaying a visual representation ofthe detected photons, and selecting a portion of the visualrepresentation based on user input according to an embodiment of thedisclosure. Blocks 1401 through 1407 correspond to blocks 1301 through1307, respectively, as described above. At block 1408, an input isreceived from a user via a user input device, such as, e.g., a computermouse, pointing device, keyboard, or touch-screen. The input may be auser-requested selection, at block 1409, of a specific spectra and/orimage to be displayed on the display device, as discussed above.

FIG. 15 is a flow chart of a further method for detecting photons usinga FAST based spectroscopic system, displaying a visual representation ofthe detected photons, storing a second visual representation in a memorydevice, and overlaying the two visual representations according to anembodiment of the disclosure. Blocks 1501 through 1507 correspond toblocks 1301 through 1307, respectively, as described above. At block1510, a second visual representation of a spectrum and/or image may bestored in a memory device. At block 1511, the second visualrepresentation may be overlaid with a first visual representation suchas the visual representation discussed above with respect to block 1307in FIG. 13. This overlaying of visual representations may be performedwith or without user input, such as the user input described withrespect to blocks 1408 and/or 1409 in FIG. 14.

The above description is not intended and should not be construed to belimited to the examples given but should be granted the full breadth ofprotection afforded by the appended claims and equivalents thereto.Although the disclosure is described using illustrative embodimentsprovided herein, it should be understood that the principles of thedisclosure are not limited thereto and may include modification theretoand permutations thereof.

1. In a system for detecting photons from a sample wherein a firstportion of said sample is illuminated by first photons from a photonsource whereby an interaction between said first photons and said sampleproduces second photons, and wherein said second photons are received bysubstantially all of the optical fibers in a fiber array spectraltranslator, and wherein said second photons are directed by saidsubstantially all of the optical fibers in said fiber array spectraltranslator to a photon detector which detects said second photons, theimprovement comprising increasing the confocality of said system bydirecting only said second photons in a predetermined group of saidplural fibers to said photon detector, wherein each fiber in saidpredetermined group is associated with a predetermined different secondportion of said sample wherein each of said predetermined differentsecond portions is smaller than said first portion.
 2. The system ofclaim 1 wherein a number of fibers in said predetermined group is lessthan or equal to twenty-five percent of a total number of fibers in saidfiber array spectral translator.
 3. The system of claim 1 wherein anumber of fibers in said predetermined group is less than or equal toten percent of a total number of fibers in said fiber array spectraltranslator.
 4. The system of claim 1 wherein a number of fibers in saidpredetermined group is less than or equal to one percent of a totalnumber of fibers in said fiber array spectral translator.
 5. The systemof claim 1 wherein ones of said predetermined different second portionsoverlap.
 6. The system of claim 1 wherein said photon detector isselected from the group consisting of: charge-coupled device (“CCD”),complementary metal oxide semiconductor (“CMOS”) detector, and focalplane array sensor.
 7. The system of claim 1 wherein said photon sourceis a laser.
 8. The system of claim 1 wherein said second photons areselected from the group consisting of: fluorescence photons from saidsample, photons emitted by said sample, photons reflected from saidsample, and photons refracted by said sample.
 9. The system of claim 1wherein said second photons are scattered photons.
 10. The system ofclaim 1 wherein said second photons are Raman scattered photons.
 11. Thesystem of claim 1 wherein a number of fibers in said predetermined groupis one.
 12. A system for detecting photons from a sample, comprising: aphoton source for illuminating a first portion of a sample with firstphotons to thereby produce second photons; a fiber array spectraltranslator comprising plural fibers for receiving said second photonsand directing said second photons to said photon detector, wherein onlya first predetermined group of said plural fibers receive and directsaid second photons to said photon detector, and wherein each fiber insaid first predetermined group is associated with a predetermineddifferent second portion of said sample wherein each of saidpredetermined different second portions is smaller than said firstportion; and said photon detector for detecting said second photons. 13.The system of claim 12 wherein a number of fibers in said predeterminedgroup is less than or equal to twenty-five percent of a total number offibers in said fiber array spectral translator.
 14. The system of claim12 wherein a number of fibers in said predetermined group is less thanor equal to ten percent of a total number of fibers in said fiber arrayspectral translator.
 15. The system of claim 12 wherein a number offibers in said predetermined group is less than or equal to one percentof a total number of fibers in said fiber array spectral translator. 16.The system of claim 12 wherein ones of said predetermined differentsecond portions overlap.
 17. The system of claim 12 wherein said photondetector is selected from the group consisting of: charge-coupled device(“CCD”), complementary metal oxide semiconductor (“CMOS”) detector, andfocal plane array sensor.
 18. The system of claim 12 wherein said photonsource is a laser.
 19. The system of claim 12 wherein said secondphotons are selected from the group consisting of: fluorescence photonsfrom said sample, photons emitted by said sample, photons reflected fromsaid sample, and photons refracted by said sample.
 20. The system ofclaim 12 wherein said second photons are scattered photons.
 21. Thesystem of claim 12 wherein said second photons are Raman scatteredphotons.
 22. The system of claim 12 wherein a number of fibers in saidpredetermined group is one.
 23. The system of claim 12 wherein saidphoton source illuminates said first portion of the sample with firstphotons via a second predetermined group of said plural fibers.
 24. Thesystem of claim 23 wherein said first and second predetermined groupsare the same and said first and second portions of the sample aresubstantially the same.
 25. The system of claim 23 wherein said firstpredetermined group is a first predetermined one of said plural fibers.26. The system of claim 25 wherein said first portion is smaller thansaid second portion.
 27. The system of claim 23 wherein said secondpredetermined group is a second predetermined one of said plural fibers.28. The system of claim 27 wherein said second portion is smaller thansaid first portion.
 29. The system of claim 23 wherein said firstpredetermined group is a first predetermined one of said plural fibersand wherein said second predetermined group is a second predeterminedone of said plural fibers.
 30. The system of claim 12 furthercomprising: a display device, and a microprocessor unit operativelyconnected to said photon detector and to said display device, whereinsaid photon detector sends to said microprocessor a first signalrepresentative of said second photons, and wherein said microprocessorunit forms a second signal from said first signal and sends said secondsignal to said display device to display a first visual representationof said second signal.
 31. The system of claim 30 wherein said firstvisual representation is a spectrum.
 32. The system of claim 31 whereinsaid spectrum is a Raman spectrum.
 33. The system of claim 30 whereinsaid first visual representation is an image.
 34. The system of claim 33wherein said image is a Raman image.
 35. The system of claim 33 whereinsaid microprocessor unit runs a software program for forming said secondsignal from said first signal.
 36. The system of claim 30 wherein saiddisplay device comprises a user-input screen for a user to select aportion of said first visual representation.
 37. The system of claim 30wherein said user-input screen is a touch-screen.
 38. The system ofclaim 30 further comprising a user input device operatively connected tosaid microprocessor unit, wherein said input device enables a user toselect a portion of said first visual representation.
 39. The system ofclaim 38 wherein said user input device is a computer mouse.
 40. Thesystem of claim 30 wherein a memory stores a second visualrepresentation, and wherein said microprocessor unit further comprisescircuitry for overlaying said first visual representation on said secondvisual representation.
 41. The system of claim 40 wherein said secondvisual representation is an optical image of said sample.
 42. The systemof claim 41 wherein said second visual representation is an opticalimage of a third portion of said sample.
 43. The system of claim 42wherein said third portion of said sample is substantially the same assaid first portion of said sample.
 44. The system of claim 12 whereinsaid first photons have a wavelength selected from the group ofwavelengths consisting of: ultraviolet light, visible light, nearinfrared light, infrared light, and combinations thereof.
 45. The systemof claim 12 wherein said second photons are fluorescence emissionphotons from said sample.
 46. A method for detecting photons from asample, comprising: illuminating a first portion of a sample with firstphotons to thereby produce second photons; receiving said second photonsusing a fiber array spectral translator comprising plural fibers anddirecting said second photons to said photon detector using said fiberarray spectral translator, wherein only a first predetermined group ofsaid plural fibers receive and direct said second photons to said photondetector, and wherein each fiber in said first predetermined group isassociated with a predetermined different second portion of said samplewherein each of said predetermined different second portions is smallerthan said first portion; and detecting said second photons using saidphoton detector.
 47. The method of claim 46 wherein a number of fibersin said predetermined group is less than or equal to twenty-five percentof a total number of fibers in said fiber array spectral translator. 48.The method of claim 46 wherein a number of fibers in said predeterminedgroup is less than or equal to ten percent of a total number of fibersin said fiber array spectral translator.
 49. The method of claim 46wherein a number of fibers in said predetermined group is less than orequal to one percent of a total number of fibers in said fiber arrayspectral translator.
 50. The method of claim 46 wherein ones of saidpredetermined different second portions overlap.
 51. The method of claim46 wherein said photon detector is selected from the group consistingof: charge-coupled device (“CCD”), complementary metal oxidesemiconductor (“CMOS”) detector, and focal plane array sensor.
 52. Themethod of claim 46 wherein said photon source is a laser.
 53. The methodof claim 46 wherein said second photons are selected from the groupconsisting of: fluorescence photons from said sample, photons emitted bysaid sample, photons reflected from said sample, and photons refractedby said sample.
 54. The method of claim 46 wherein said second photonsare scattered photons.
 55. The method of claim 46 wherein said secondphotons are Raman scattered photons.
 56. The method of claim 46 whereina number of fibers in said predetermined group is one.
 57. The method ofclaim 46 wherein said photon source illuminates said first portion ofthe sample with first photons via a second predetermined group of saidplural fibers.
 58. The method of claim 57 wherein said first and secondpredetermined groups are the same and said first and second portions ofthe sample are substantially the same.
 59. The method of claim 57wherein said first predetermined group is a first predetermined one ofsaid plural fibers.
 60. The method of claim 59 wherein said firstportion is smaller than said second portion.
 61. The method of claim 57wherein said second predetermined group is a second predetermined one ofsaid plural fibers.
 62. The method of claim 61 wherein said secondportion is smaller than said first portion.
 63. The method of claim 57wherein said first predetermined group is a first predetermined one ofsaid plural fibers and wherein said second predetermined group is asecond predetermined one of said plural fibers.
 64. The method of claim46 further comprising: sending a first signal representative of saidsecond photons to a microprocessor unit, wherein said microprocessorunit forms a second signal from said first signal; sending said secondsignal from said microprocessor unit to a display device, and displayinga first visual representation of said second signal.
 65. The method ofclaim 64 wherein said first visual representation is a spectrum.
 66. Themethod of claim 65 wherein said spectrum is a Raman spectrum.
 67. Themethod of claim 64 wherein said first visual representation is an image.68. The method of claim 67 wherein said image is a Raman image.
 69. Themethod of claim 67 wherein said microprocessor unit runs a softwareprogram for forming said second signal from said first signal.
 70. Themethod of claim 64 further comprising receiving input from a user onsaid display device comprising a user-input screen, wherein said userselects a portion of said first visual representation.
 71. The method ofclaim 64 wherein said user-input screen is a touch-screen.
 72. Themethod of claim 64 further comprising a receiving input from a userinput device operatively connected to said microprocessor unit, whereinsaid user selects a portion of said first visual representation.
 73. Themethod of claim 72 wherein said user input device is a computer mouse.74. The method of claim 64 further comprising storing a second visualrepresentation in a memory device, and overlaying said first visualrepresentation on said second visual representation.
 75. The method ofclaim 74 wherein said second visual representation is an optical imageof said sample.
 76. The method of claim 75 wherein said second visualrepresentation is an optical image of a third portion of said sample.77. The method of claim 76 wherein said third portion of said sample issubstantially the same as said first portion of said sample.
 78. Themethod of claim 46 wherein said first photons have a wavelength selectedfrom the group of wavelengths consisting of: ultraviolet light, visiblelight, near infrared light, infrared light, and combinations thereof.79. The method of claim 46 wherein said second photons are fluorescenceemission photons from said sample.